The candidate, Gil Redelman-Sidi, a physician-scientist in Memorial Sloan Kettering Cancer Center with a clinical and research interest in the interface between infection and cancer, seeks to achieve a career as a researcher in the field of the treatment of bladder cancer with microbial biotherapies. This proposal describes a 5-year training program that will provide the candidate with the knowledge base and technical skills necessary to achieve this goal. In addition to intensive laboratory-based experimental training, the training program will include regular meetings with an advisory committee of experts in the relevant fields, active involvement in conferences and meetings, and formal didactics that will include a full course in cell signaling and development, cell cycle control, immunology and cancer biology. At the end of the period of support, the candidate will be poised to undertake a career as an independent physician-scientist. This revised proposal focuses on the mechanisms of action of BCG for the treatment of bladder cancer, one of the most common cancers worldwide. The standard therapy for patients with early-stage bladder cancer (not invading the muscular layer of the bladder wall) is tumor resection followed by bladder instillation therapy with BCG, a live bacterium originally developed as a vaccine against tuberculosis. BCG effectively eradicates residual tumor and lowers risk of recurrence. Nevertheless, bladder cancer recurs or progresses in over a third of BCG-treated patients. Despite being in use for nearly four decades, the mechanism by which BCG acts remains poorly understood, and there is no way to reliably predict clinical response. Bladder cancer cells can internalize BCG, but until recently the pathway of uptake was not known. The candidate has shown that this pathway is macropinocytosis. Furthermore, the candidate has shown that the ability of bladder cancer cells to internalize BCG depends on the presence of certain oncogenic (tumor- causing) mutations. Recently, the candidate, through a series of whole-genome screens, has identified numerous additional genes, in bladder cancer cells and in BCG, that determine the internalization or survival of BCG in bladder cancer cells. In this project, the candidate will extend these prior findings to an in vivo model of bladder cancer progression and BCG response. The specific aims are as follows: (1) Establish the relationship between BCG internalization by bladder cancer cells and clinical response to BCG therapy in murine models of bladder cancer by testing clinical response to BCG therapy in mice implanted with tumors in which pathways implicated in macropinocytotic BCG uptake, including the Wnt pathway (identified in our whole-genome screens as stimulating BCG uptake and involved in macropinocytosis), have been manipulated through genetic or pharmacologic means. (2) Test, in cultured cells and in the murine bladder cancer model, response to recombinant BCG strains lacking or overexpressing the serine/threonine protein kinases pknG and pknL, both of which we have identified as determinants of BCG uptake and/or survival within bladder cancer cells. If successful, this project could lead to clinical trials in patients receiving BCG for bladder cancer Potential clinical benefits of these studies include: (1) development of assays to predict a patient's response to BCG therapy based on phenotypic or genotypic characteristics of his/her tumor; testing methods to improve BCG efficacy, including (2) through co-administration of BCG with pharmacologic agents that increase its uptake by bladder cancer cells and (3) by constructing recombinant BCG strains with improved therapeutic efficacy and improved safety.